In order to introduce a gene to a cultured cell or living tissue for gene function analysis or gene therapy, many viral methods and non-viral methods have been developed (Mulligan, Science, vol. 260, p. 926-932, (1993); Ledley, Human Gene Therapy, vol. 6, p. 1129-1144 (1995)). Generally, in introducing genes into cells, viral methods are effective. However, methods using viral vectors involve possible introduction of a gene derived from the parent virus and expression thereof, immunogenicity, and possible modification of the host genome structure, posing a problem with safety. Meanwhile, many of non-viral methods, which use liposomes and the like, tend to be inferior to viral vectors in terms of the efficiency of gene introduction to a cultured cell or living tissue, though the cytotoxicity and immunogenicity thereof are lower than those of viral methods.
It is thought that viruses gradually change the functions thereof to achieve coexistence with the host by repeating mutations, even though they are initially fatally pathogenic to the host, whereby they can survive. Therefore, many mutants, even in a single virus, occur in the natural world; it can be said that investigations thereof have led to the elucidation of the functions possessed by viruses at the molecular level. This fact is not only valuable from the viewpoint of basic biology, but also has been emphasized from the viewpoint of medical aspects such as development of antiviral vaccines. Isolation of a spontaneously emerging virus mutant strain takes a long time, and a desired mutant strain can be obtained only by chance.
HVJ (hemagglutinating virus of Japan; Sendai virus) is a virus belonging to the paramyxovirus family, having an envelope on the surface of which hemagglutinin and neuraminidase are present. HVJ has (−)-stranded RNA in the genome thereof; after infection of HVJ to host cells, (+)-stranded RNA is replicated from the (−) strand, from which a large amount of virus protein is produced, resulting in the formation of virus particles, which in turn bud to produce new virus particles. HVJ attracted attention as a fusogen for Ehrlich's tumor cells (Okada, Biken Journal, 1, p. 103-110, (1958)); the cell membrane fusion activity thereof (hereinafter, fusion activity) has been analyzed, and the utilization thereof as a gene introduction vector has been investigated. In the fusion, first, HN (hemagglutinating) protein recognizes acetyl-type sialic acid on the cell surface and decomposes the sugar chain thereof by the activity of sialidase, and then F (fusion) protein enters the lipid bilayer to induce the fusion. As such, HN protein possesses hemagglutination activity and hemolytic activity, and, when administered into the blood, causes a transient reduction in blood coagulation capacity. This virus has also been developed as a vector for gene transfection and drug delivery, but it is also anticipated that even if HVJ incorporating a target molecule is prepared, the specificity thereof will be lessened in the presence of HN protein.
Therefore, there is a demand for the development of a highly safe viral vector wherein the toxicity of receptor proteins such as HN protein has been ameliorated.
HVJ (hemagglutinating virus of Japan; Sendai virus) is also well known as a mouse parainfluenza virus that causes cell fusion; utilizing the function, gene transfection vectors such as recombinant Sendai viral vectors, HVJ envelope vectors, and HVJ-liposomes, and drug delivery systems, have been developed. In the fusion, HN (hemagglutinating) protein recognizes acetyl-type sialic acid on the cell surface, decomposes the sugar chain thereof by the activity of sialidase, and then F (fusion) protein enters the lipid bilayer to induce the fusion. Therefore, almost all cells having sialic acid can be subjects of the fusion. HVJ is highly versatile for a vector, but on the other hand it has no specificity and is not suitable for the introduction of cytotoxic molecules. One of the major problems to be solved in improving a vector is to enhance the specificity, specifically to enable target introduction. The present inventors' group has already succeeded in preparing liposomes using some lipids having an amino group, binding a chemically modified antibody molecule to the amino group using a crosslinking agent to prepare an immunoliposome, and fusing this liposome with inactivated HVJ to develop an HVJ-immunoliposome (Tomita, N et al, J. Gene Medicine, vol. 4, p. 527-535 (2002)). When an HVJ-immunoliposome prepared using a monoclonal antibody that recognizes the Thy-1 antigen of rat kidney mesangium cells was injected from a peripheral blood vessel, the HVJ-liposome, which otherwise gathers in the reticuloendothelial systems of the liver and spleen, accumulated in the mesangium of both kidneys, making it possible to introduce a fluorescent oligo-nucleic acid into 90% of the glomeruli. This fact suggests that even in the presence of HN protein, provided that a target molecule is inserted, and is first recognized and binds to a specific cell, fusion occurs there and enables specific introduction. However, the method of binding a protein like this antibody to a liposome is so complex that skills are required to obtain constant results.
Therefore, there is a demand for the development of a viral vector of high tissue and cell specificity, and a method of obtaining the viral vector conveniently and stably. For example, in Hallak et al., Cancer Res, vol. 65(12), p. 5292-5300 (2005), it is reported that in an attempt to fuse the M28L echistatin molecule, which binds to integrin αvβ3, to the C-terminus of the H protein of measles virus, they modified the virus genome and prepared a virus that targets cancer cells having integrin αvβ3. Furthermore, attempts have been made to modify various viral vectors to prepare targeting viral vectors (JP-T-2001-515493, JP-A-2002-320475, JP-T-2005-532075, JP-A-2002-330774).